Certal Residual DNA Detection Kits

For sensitive probe-based detection of residual CHO host cell DNA, including an internal control

Low limit of quantification with excellent reproducibility

High linear range

Reliable and precise internal control

Optimized protocols for a broad range of real-time cyclers

Complete kits including internal control and positive control

When working with biopharmaceuticals, it is essential to evaluate the final therapeutic material to ensure that it is free of contaminating substances, such as residual DNA from the host cell. Certal Residual DNA Detection Kits are designed for sensitive and rapid real-time PCR quantification of residual host cell genomic DNA using dual-labeled hydrolysis (also known as TaqMan®) probes. The Certal CHO Detection Kit contains reagents for duplex real-time PCR detection of CHO residual host cell DNA, plus the Certal Internal Control. The kit also includes the Certal Residual DNA Positive Control, which verifies the functionality of the PCR assay in the absence of a signal from CHO host cell DNA, and 2 formats of ROX passive reference dye in different concentrations, enabling use on virtually any real-time instrument.

The Certal Residual DNA Detection Kits are intended for molecular biology applications in pharmaceutical product testing. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Reliable detection of the QIAGEN Internal Control.|Certal Residual DNA Detection Kit procedure.|Sensitive and reliable detection of CHO genomic DNA on the Rotor-Gene Q.|Low limit of detection.|Sensitive and reliable detection on a wide range of cyclers.|Highly sensitive and reliable detection.|

Primers and probe for the target (CHO) assay and the internal control assay are added to the Certal ResDNA Master Mix. Once internal control template and sample template is added and reactions are distributed, they can be run using a wide range of cyclers. Optionally, positive CHO controls can be tested, and UNG may be used to control for carryover contamination of PCR products.|Primers and probe for the target (CHO) assay and the internal control assay are added to the Certal ResDNA Master Mix. Once internal control template and sample template is added and reactions are distributed, they can be run using a wide range of cyclers. Optionally, positive CHO controls can be tested, and UNG may be used to control for carryover contamination of PCR products.|The reliability of the Certal Residual DNA Assay on the Rotor-Gene Q is demonstrated by evenly spaced amplification plots for the target signal and stable CT values for the internal control (inset), which indicate that sensitive detection of the internal control is not affected by the wide range of CHO genomic DNA concentration.|The limit of detection (LOD), defined as the lowest concentration with 100% positive amplification result for 24 replicates, was determined to be 1 fg using the Certal CHO Detection Kit Assay. The limit of quantification (LOQ), defined as the lowest concentration of a calculated unknown within ±0.5 CT of the standard value, is 10 fg.|Target DNA ranging from 3 fg to 30 ng was analyzed using the Certal CHO Detection Kit on a wide range of commonly used cyclers. The high linear range of detection demonstrates the reliability and adaptability of Certal assays on several different qPCR cyclers.|CHO DNA (0.3 fg to 300 ng) was analyzed using the Certal CHO Detection Kit on the Applied Biosystems ABI 7900 HT (384-well format). Highly sensitive detection was achieved with a low limit of quantification and a high linear range of detection, ranging from 10 fg to 300 ng. Error bars, demonstrating the reliability of the reaction, each represent ±1 SD of 4 replicates and are smaller than data points where not visible. LOQ: Limit of quantification, defined as the lowest concentration of a calculated unknown within ±0.5 CTof the standard value. This was determined experimentally: the mean of the unknowns were not permitted to deviate more than 0.5 CT from the mean of the standard curve. LOD: Limit of detection, defined as the lowest concentration with 100% positive amplification result for 24 replicates.|

For high process safety, Certal Residual DNA Detection Kits include the QIAGEN Internal Control, which is specifically designed to enable the correct interpretation of negative results and to not interfere with the target assay. The QIAGEN Internal Control is reliably detected when coamplified with genomic DNA (see figure "Reliable detection of the QIAGEN Internal Control").

Principle

To ensure that contaminating substances from the host cell line used to produce biopharmaceuticals are absent from final therapeutic material, Certal Residual DNA Detection Kits deliver a predesigned assay to specifically detect CHO host cell DNA. Each Certal Residual DNA Assay consists of an optimized primer pair and a FAM labeled hydrolysis probe for reliable detection of residual CHO genomic DNA. Human background DNA and Certal Internal Control DNA will not interfere with the Certal Residual DNA Assay.

The premixed Certal Internal Control Assay contains a forward and reverse primer and a MAX labeled hydrolysis probe for the detection of Certal Internal Control DNA. It has been specifically optimized to prevent interference with Certal Residual DNA Assays. The Certal Residual DNA Positive Control enables the correct interpretation of negative results by testing the functionality of the amplification reaction itself.

Certal Residual DNA Detection Kits enable sensitive real-time PCR using sequence-specific probes for the detection of host-cell DNA, including an internal control. The kits can be used on a wide range of real-time cyclers, including cyclers from QIAGEN, Applied Biosystems, Bio-Rad, Roche, and Agilent.